Methods of modifying cell structure and remodeling tissue

ABSTRACT

Disclosed is a method of modifying cell structure which includes: increasing the intracellular concentration of biliverdin reductase, or a fragment or variant thereof, in a mammalian cell under conditions effective to modify the structure of the mammalian cell. Also disclosed are methods of performing in vivo tissue remodeling in a mammal and repairing a damaged organ or organ system, both of which include delivering biliverdin reductase, or fragments or variants thereof, to one or more cells present at a site of tissue remodeling in a mammal, wherein said delivering increases the intracellular concentration of biliverdin reductase, or fragments or variants thereof, under conditions effective to modify the structure of the one or more cells at the site of tissue remodeling, thereby remodeling the tissue containing the one or more cells.

This application is a divisional application of U.S. patent applicationSer. No. 10/045,545 filed Jan. 14, 2002, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/261,500, filed Jan. 12,2001, each of which is hereby incorporated by reference in its entirety.

This work was supported by the U.S. National Institutes of Health underGrant Nos. ES04066 and ES04391. The U.S. Government may have certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods of modifying cell structure andremodeling tissue which involve modulating the intracellular levels ofbiliverdin reductase or active fragments or variants thereof.

BACKGROUND OF THE INVENTION

Biliverdin reductase (“BVR”) catalyzes reduction of the γ-meso bridge ofbiliverdin, an open tetrapyrrole, to produce bilirubin (Singleton etal., J. Biol. Chem. 240: 47890-4789 (1965); Tenhunen et al.,Biochemistry 9:298-303 (1970); Colleran et al., Biochem J. 119:16P-19P(1970); Kutty et al., J. Biol. Chem. 256:3956-3962 (1981); Buldain etal., Eur. J. Biochem. 156:179-184 (1986); Noguchi et al., Biochem J.86:833-839 (1989)). In mammals, the oxidative cleavage of heme iscatalyzed by the heme oxygenase system (Maines, Ann. Rev. Pharmacol.Toxicol. 37:517-554 (1997)). Because open tetrapyrroles are generallybelieved to be devoid of biological functions, the enzymes that catalyzetheir formation have not traditionally been in the main stream ofresearch activity. In plants, however, biliverdin analogues,phytochromobilins, function in photoregulatory capacity (Terry et al.,J. Biol. Chem. 266:22215-22221 (1991); Cornejo et al., J. Biol. Chem.267:14790-14798 (1992)). Molecular cloning and biochemical analyses haveshown that the enzyme, which in human is a 296 residue polypeptide, ishighly conserved both at its primary structure and at its uniquecatalytic properties (Fakhrai et al., J. Biol. Chem. 267:4023-4029(1992); McCoubrey et al., Eur. J Biochem. 222:597-603 (1994); McCoubreyet al., Gene 160:235-240 (1995); Maines et al., Eur. J. Biochem.235:372-381 (1996)). BVR is the only enzyme described to date with dualpH/dual adenine nucleotide cofactor requirements (Kutty et al., J. Biol.Chem. 256:3956-3962 (1981); Fakhrai et al., J. Biol. Chem. 267:4023-4029(1992); Maines et al., Eur. J. Biochem. 235:372-381 (1996); Huang etal., J. Biol. Chem. 264:7844-7849 (1989)). The reductase uses NADH inthe acidic range (optimum range ˜pH 6.0-6.7), whereas NADPH is utilizedin the basic range (optimum range ˜pH 8.5-8.7). BVR, which is a zincmetalloprotein (Maines et al., Eur. J. Biochem. 235:372-381 (1996)),possesses a HCX₁₀CH or HCX₁₀CC motif in the carboxy terminal third ofthe protein, which is similar to the zinc binding motif of proteinkinase C (Hubbard et al., Science 254:1776-1779 (1991)) and may be thesite of interaction of BVR with zinc.

BVR was previously thought to be simply a house-keeping enzyme found inmost mammalian cells in excess of, or in disproportionate levels to,heme oxygenase isozymes (Ewing et al., J. Neurochem. 61:1015-1023(1993)). Yet it has the above-noted noted unique and uncommonproperties. Examination of the primary structure of human BVR, whichrecently became available (Maines et al., Eur. J. Biochem. 235:372-381(1996)), revealed the presence of consensus sequences that are conservedin protein kinases, the most notable one being the GXGXXG motif near theN terminus of the protein that is found invariably in all kinases (Kampset al., Nature 310:589-592 (1984); Hunter et al., Ann. Rev. Biochem.54:897-930 (1985); Schlessinger, Trend. Biochem. Sci. 13:443-447 (1988);Hanks et al., Science 241:42-52 (1988); Yarden et al., Annu. Rev.Biochem. 57:443-478 (1988); Hanks et al., Methods Enzymol. 200:38-62(1991)). A valine residue is present in BVR just 2 positions downstreamfrom the last glycine of this motif. A valine residue is invariant atthe corresponding position, as in BVR, in the family of kinases thatphosphorylate G-protein coupled receptors (Garcia-Bustos et al.,Biochim. Biophys. ACTA 1071:83-101 (1991)). Database search results alsoidentified additional similarities with PKGs, including a cluster ofcharged residues (KRNR) in the carboxy terminus of BVR. Such clustersare a characteristic of the nuclear localization signal (“NLS”)(Garcia-Bustos et al., Biochim. Biophys. ACTA 1071:83-101 (1991)).

Although BVR has previously been identified as exhibiting protectiveeffects against oxidative stress and as sharing characteristics withknown kinases (see U.S. patent application Ser. No. 09/606,129 toMaines, filed Jun. 28, 2000), it is unclear the extent to which BVR isimplicated in cellular repair mechanisms. The present invention isdirected to overcoming the above-identified deficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method of modifyingcell structure which includes: increasing the intracellularconcentration of biliverdin reductase, or a fragment or variant thereof,in a mammalian cell under conditions effective to modify the structureof the mammalian cell.

A second aspect of the present invention relates to a method of in vivotissue remodeling in a mammal which includes: delivering biliverdinreductase, or fragments or variants thereof, to one or more cellspresent at a site of tissue remodeling in a mammal, wherein saiddelivering increases the intracellular concentration of biliverdinreductase, or fragments or variants thereof, under conditions effectiveto modify the structure of the one or more cells at the site of tissueremodeling, thereby remodeling the tissue containing the one or morecells.

A third aspect of the present invention relates to a method of repairinga damaged organ or organ system by performing the in vivo tissuemodeling of the present invention, where the site of tissue remodelingis within the damaged organ or organ system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 show different images of HeLa cells transfected with human BVRDNA. Differences between untransfected cells (normal morphology) andtransfected cells (immunostained) is readily apparent. The transfectedcells show remarkable enlargement, altered cellular morphology includingfilopodia, and the appearance of spikes which are clearly observed.

DETAILED DESCRIPTION OF THE INVENTION

The methods of modifying cell structure and remodeling tissue accordingto the present invention involve modulating the intracellular levels ofbiliverdin reductase (“BVR”) or active fragments thereof. Modulatingintracellular levels of BVR can be achieved using known recombinanttechniques directed to cells or tissues to be affected, as describedbelow, or by using known protein delivery techniques for delivering BVRinto cells or tissues to be affected.

One form of human biliverdin reductase (“hBVR”) has an amino acidsequence corresponding to SEQ. ID. No. 1 as follows: Met Asn Ala Glu ProGlu Arg Lys Phe Gly Val Val Val Val Gly Val 1 5 10 15 Gly Arg Ala GlySer Val Arg Met Arg Asp Leu Arg Asn Pro His Pro 20 25 30 Ser Ser Ala PheLeu Asn Leu Ile Gly Phe Val Ser Arg Arg Glu Leu 35 40 45 Gly Ser Ile AspGly Val Gln Gln Ile Ser Leu Glu Asp Ala Leu Ser 50 55 60 Ser Gln Glu ValGlu Val Ala Tyr Ile Cys Ser Glu Ser Ser Ser His 65 70 75 80 Glu Asp TyrIle Arg Gln Phe Leu Asn Ala Gly Lys His Val Leu Val 85 90 95 Glu Tyr ProMet Thr Leu Ser Leu Ala Ala Ala Gln Glu Leu Trp Glu 100 105 110 Leu AlaGlu Gln Lys Gly Lys Val Leu His Glu Glu His Val Glu Leu 115 120 125 LeuMet Glu Glu Phe Ala Phe Leu Lys Lys Glu Val Val Gly Lys Asp 130 135 140Leu Leu Lys Gly Ser Leu Leu Phe Thr Ser Asp Pro Leu Glu Glu Asp 145 150155 160 Arg Phe Gly Phe Pro Ala Phe Ser Gly Ile Ser Arg Leu Thr Trp Leu165 170 175 Val Ser Leu Phe Gly Glu Leu Ser Leu Val Ser Ala Thr Leu GluGlu 180 185 190 Arg Lys Glu Asp Gln Tyr Met Lys Met Thr Val Cys Leu GluThr Glu 195 200 205 Lys Lys Ser Pro Leu Ser Trp Ile Glu Glu Lys Gly ProGly Leu Lys 210 215 220 Arg Asn Arg Tyr Leu Ser Phe His Phe Lys Ser GlySer Leu Glu Asn 225 230 235 240 Val Pro Asn Val Gly Val Asn Lys Asn IlePhe Leu Lys Asp Gln Asn 245 250 255 Ile Phe Val Gln Lys Leu Leu Gly GlnPhe Ser Glu Lys Glu Leu Ala 260 265 270 Ala Glu Lys Lys Arg Ile Leu HisCys Leu Gly Leu Ala Glu Glu Ile 275 280 285 Gln Lys Tyr Cys Cys Ser ArgLys 290 295

Heterologous expression and isolation of hBVR is described in Maines etal., Eur. J. Biochem. 235(1-2):372-381 (1996); Maines et al., Arch.Biochem. Biophys. 300(1):320-326 (1993), each of which is herebyincorporated by reference in its entirety. The DNA molecule encodingthis form of hBVR has a nucleotide sequence corresponding to SEQ. ID.No. 2 as follows: ggggtggcgc ccggagctgc acggagagcg tgcccgtcag tgaccgaagaagagaccaag 60 atgaatgcag agcccgagag gaagtttggc gtggtggtgg ttggtgttggccgagccggc 120 tccgtgcgga tgagggactt gcggaatcca cacccttcct cagcgttcctgaacctgatt 180 ggcttcgtgt cgagaaggga gctcgggagc attgatggag tccagcagatttctttggag 240 gatgctcttt ccagccaaga ggtggaggtc gcctatatct gcagtgagagctccagccat 300 gaggactaca tcaggcagtt ccttaatgct ggcaagcacg tccttgtggaataccccatg 360 acactgtcat tggcggccgc tcaggaactg tgggagctgg ctgagcagaaaggaaaagtc 420 ttgcacgagg agcatgttga actcttgatg gaggaattcg ctttcctgaaaaaagaagtg 480 gtggggaaag acctgctgaa agggtcgctc ctcttcacat ctgacccgttggaagaagac 540 cggtttggct tccctgcatt cagcggcatc tctcgactga cctggctggtctccctcttt 600 ggggagcttt ctcttgtgtc tgccactttg gaagagcgaa aggaagatcagtatatgaaa 660 atgacagtgt gtctggagac agagaagaaa agtccactgt catggattgaagaaaaagga 720 cctggtctaa aacgaaacag atatttaagc ttccatttca agtctgggtccttggagaat 780 gtgccaaatg taggagtgaa taagaacata tttctgaaag atcaaaatatatttgtccag 840 aaactcttgg gccagttctc tgagaaggaa ctggctgctg aaaagaaacgcatcctgcac 900 tgcctggggc ttgcagaaga aatccagaaa tattgctgtt caaggaagtaagaggaggag 960 gtgatgtagc acttccaaga tggcaccagc atttggttct tctcaagagttgaccattat 1020 ctctattctt aaaattaaac atgttgggga aacaaaaaaa aaaaaaaaaa1070The open reading frame which encodes hBVR of SEQ. ID. No. 1 extends fromnt 1 to nt 888.

Another form of hBVR has an amino acid sequence according to SEQ. ID.No. 3 as follows: Met Asn Thr Glu Pro Glu Arg Lys Phe Gly Val Val ValVal Gly Val 1 5 10 15 Gly Arg Ala Gly Ser Val Arg Met Arg Asp Leu ArgAsn Pro His Pro 20 25 30 Ser Ser Ala Phe Leu Asn Leu Ile Gly Phe Val SerArg Arg Glu Leu 35 40 45 Gly Ser Ile Asp Gly Val Gln Gln Ile Ser Leu GluAsp Ala Leu Ser 50 55 60 Ser Gln Glu Val Glu Val Ala Tyr Ile Cys Ser GluSer Ser Ser His 65 70 75 80 Glu Asp Tyr Ile Arg Gln Phe Leu Asn Ala GlyLys His Val Leu Val 85 90 95 Glu Tyr Pro Met Thr Leu Ser Leu Ala Ala AlaGln Glu Leu Trp Glu 100 105 110 Leu Ala Glu Gln Lys Gly Lys Val Leu HisGlu Glu His Val Glu Leu 115 120 125 Leu Met Glu Glu Phe Ala Phe Leu LysLys Glu Val Val Gly Lys Asp 130 135 140 Leu Leu Lys Gly Ser Leu Leu PheThr Ala Gly Pro Leu Glu Glu Glu 145 150 155 160 Arg Phe Gly Phe Pro AlaPhe Ser Gly Ile Ser Arg Leu Thr Trp Leu 165 170 175 Val Ser Leu Phe GlyGlu Leu Ser Leu Val Ser Ala Thr Leu Glu Glu 180 185 190 Arg Lys Glu AspGln Tyr Met Lys Met Thr Val Cys Leu Glu Thr Glu 195 200 205 Lys Lys SerPro Leu Ser Trp Ile Glu Glu Lys Gly Pro Gly Leu Lys 210 215 220 Arg AsnArg Tyr Leu Ser Phe His Phe Lys Ser Gly Ser Leu Glu Asn 225 230 235 240Val Pro Asn Val Gly Val Asn Lys Asn Ile Phe Leu Lys Asp Gln Asn 245 250255 Ile Phe Val Gln Lys Leu Leu Gly Gln Phe Ser Glu Lys Glu Leu Ala 260265 270 Ala Glu Lys Lys Arg Ile Leu His Cys Leu Gly Leu Ala Glu Glu Ile275 280 285 Gln Lys Tyr Cys Cys Ser Arg Lys 290 295This hBVR sequence is reported at Komuro et al., NCBI Accession No.G02066, direct submission to the EMBL Data Library (1998), which ishereby incorporated by reference in its entirety. Differences betweenthe hBVR of SEQ. ID. No. 1 and the hBVR of SEQ. ID. No. 3 are at aaresidues 3, 154, 155, and 160. Thus, residue 3 can be either alanine orthreonine, residue 154 can be either alanine or serine, residue 155 canbe either aspartic acid or glycine, and residue 160 can be eitheraspartic acid or glutamic acid.

One form of rat biliverdin reductase (“rBVR”) has an amino acid sequencecorresponding to SEQ. ID. No. 4 as follows: Met Asp Ala Glu Pro Lys ArgLys Phe Gly Val Val Val Val Gly Val  1               5                  10                  15 Gly Arg AlaGly Ser Val Arg Leu Arg Asp Leu Lys Asp Pro Arg Ser             20                  25                  30 Ala Ala Phe LeuAsn Leu Ile Gly Phe Val Ser Arg Arg Glu Leu Gly         35                  40                  45 Ser Leu Asp Glu ValArg Gln Ile Ser Leu Glu Asp Ala Leu Arg Ser     50                  55                  60 Gln Glu Ile Asp Val AlaTyr Ile Cys Ser Glu Ser Ser Ser His Glu 65                  70                  75                  80 Asp TyrIle Arg Gln Phe Leu Gln Ala Gly Lys His Val Leu Val Glu                 85                  90                  95 Tyr Pro MetThr Leu Ser Phe Ala Ala Ala Gln Glu Leu Trp Glu Leu            100                 105                 110 Ala Ala Gln LysGly Arg Val Leu His Glu Glu His Val Glu Leu Leu        115                 120                 125 Met Glu Glu Phe GluPhe Leu Arg Arg Glu Val Leu Gly Lys Glu Leu    130                 135                 140 Leu Lys Gly Ser Leu ArgPhe Thr Ala Ser Pro Leu Glu Glu Glu Arg145                 150                 155                 160 Phe GlyPhe Pro Ala Phe Ser Gly Ile Ser Arg Leu Thr Trp Leu Val                165                 170                 175 Ser Leu PheGly Glu Leu Ser Leu Ile Ser Ala Thr Leu Glu Glu Arg            180                 185                 190 Lys Glu Asp GlnTyr Met Lys Met Thr Val Gln Leu Glu Thr Gln Asn        195                 200                 205 Lys Gly Leu Leu SerTrp Ile Glu Glu Lys Gly Pro Gly Leu Lys Arg    210                 215                 220 Asn Arg Tyr Val Asn PheGln Phe Thr Ser Gly Ser Leu Glu Glu Val225                 230                 235                 240 Pro SerVal Gly Val Asn Lys Asn Ile Phe Leu Lys Asp Gln Asp Ile                245                 250                 255 Phe Val GlnLys Leu Leu Asp Gln Val Ser Ala Glu Asp Leu Ala Ala            260                 265                 270 Glu Lys Lys ArgIle Met His Cys Leu Gly Leu Ala Ser Asp Ile Gln        275                 280                 285 Lys Leu Cys His GlnLys Lys     290                 295

Heterologous expression and isolation of rBVR is described in Fakhrai etal., J. Biol. Chem. 267(6):4023-4029 (1992), which is herebyincorporated by reference in its entirety. The rBVR of SEQ. ID. No. 4shares about 82% aa identity to the hBVR of SEQ. ID. No. 1, withvariations in aa residues being highly conserved. The DNA moleculeencoding this form of rBVR has a nucleotide sequence corresponding toSEQ. ID. No. 5 as follows: ggtcaacagc taagtgaagc catatccata gagagtttgtgccagtgccc caagatcctg 60 aacctctgtc tgtcttcgga cactgactga agagaccgagatggatgccg agccaaagag 120 gaaatttgga gtggtagtgg ttggtgttgg cagagctggctcggtgaggc tgagggactt 180 gaaggatcca cgctctgcag cattcctgaa cctgattggatttgtgtcca gacgagagct 240 tgggagcctt gatgaagtac ggcagatttc tttggaagatgctctccgaa gccaagagat 300 tgatgtcgcc tatatttgca gtgagagttc cagccatgaagactatatac ggcagtttct 360 gcaggctggc aagcatgtcc tcgtggaata ccccatgacactgtcatttg cggcggccca 420 ggagctgtgg gagctggccg cacagaaagg gagagtcctgcatgaggagc acgtggaact 480 cttgatggag gaattcgaat tcctgagaag agaagtgttggggaaagagc tactgaaagg 540 gtctcttcgc ttcacagcta gcccactgga agaagagagatttggcttcc ctgcgttcag 600 cggcatttct cgcctgacct ggctggtctc cctcttcggggagctttctc ttatttctgc 660 caccttggaa gagcgaaaag aggatcagta tatgaaaatgaccgtgcagc tggagaccca 720 gaacaagggt ctgctgtcat ggattgaaga gaaagggcctggcttaaaaa gaaacagata 780 tgtaaacttc cagttcactt ctgggtccct ggaggaagtgccaagtgtag gggtcaataa 840 gaacattttc ctgaaagatc aggatatatt tgttcagaagctcttagacc aggtctctgc 900 agaggacctg gctgctgaga agaagcgcat catgcattgcctggggctgg ccagcgacat 960 ccagaagctt tgccaccaga agaagtgaag aggaagcttcagagacttct gaagggggcc 1020 agggtttggt cctatcaacc attcaccttt agctcttacaattaaacatg tcagataaac 1080 a 1081The open reading frame which encodes rBVR of SEQ. ID. No. 4 extends fromnt 1 to nt 885.

By way of example, hBVR of SEQ. ID. No. 1 is characterized by a numberof functional domains, including putative and/or demonstratedphosphorylation sites from aa 15 to 20, aa 21 to 23, aa 44 to 46 or 47,aa 49 to 54, aa 58 to 61, aa 64 to 67, aa 78 to 81, aa 79 to 82, aa 189to 192, aa 207 to 209, aa 214 to 217, aa 222 to 227, aa 236 to 241, aa245 to 250, aa 267 to 269 or 270, and aa 294 to 296; a basic N-terminaldomain characterized by aa 6 to 8; a hydrophobic domain characterized byaa 9 to 14 (FXVVVV, SEQ. ID. No. 6); a nucleotide binding domaincharacterized by aa 15 to 20 (GXGXXG, SEQ. ID. No. 7); an oxidoreductasedomain characterized by aa 90 to 97 (AGKHVLVE, SEQ. ID. No. 8); aleucine zipper spanning aa 129 to 157 (LX₆LX₆KX₆LX₆L, SEQ. ID. No. 9);several kinase motifs, including aa 44 to 46 (SRR, SEQ. ID. No. 10), aa147 to 149 (KGS, SEQ. ID. No. 11) and aa 162 to 164 (FGX, SEQ. ID. No.12); a nuclear localization signal spanning aa 222 to 228 (GLKRNRY, SEQ.ID. No. 13); a myristylation site spanning aa 221 to 225 (PGLKR, SEQ.ID. No. 14); a zinc finger domain spanning aa 280 to 293 (HCX₁₀CC, SEQ.ID. No. 15); and substrate binding domains including, withoutlimitation, a protein kinase C (“PKC”) enhancing domain spanning aa 275to 281 (KKRIXHC, SEQ. ID. No. 16) and a PKC inhibiting domain spanningaa 289 to 296 (QKXCXXXK, SEQ. ID. No. 17). By way of sequence comparisonand, in consideration of conserved substitutions, hBVR of SEQ. ID. No. 3and rBVR of SEQ. ID. No. 4 include similar functional domains. Forexample rBVR includes an identical hydrophobic domain, an identicalnucleotide binding domain, an identical oxidoreductase domain, aconserved leucine zipper domain (with residue variations between L and Kresidues), identical or conserved kinase motifs, an identical nuclearlocalization signal, an identical myristylation site, a conserved zincfinger domain (with terminal C residue replaced by H), a conserved PKCenhancing domain, and a conserved PKC inhibiting domain.

DNA molecules encoding a BVR protein or polypeptide can also include aDNA molecule that hybridizes under stringent conditions to the DNAmolecule having a nucleotide sequence of SEQ. ID. No. 2 or SEQ. ID. No.5. An example of suitable stringency conditions is when hybridization iscarried out at a temperature of about 37° C. using a hybridizationmedium that includes 0.9M sodium citrate (“SSC”) buffer, followed bywashing with 0.2×SSC buffer at 37° C. Higher stringency can readily beattained by increasing the temperature for either hybridization orwashing conditions or increasing the sodium concentration of thehybridization or wash medium. Nonspecific binding may also be controlledusing any one of a number of known techniques such as, for example,blocking the membrane with protein-containing solutions, addition ofheterologous RNA, DNA, and SDS to the hybridization buffer, andtreatment with RNase. Wash conditions are typically performed at orbelow stringency. Exemplary high stringency conditions include carryingout hybridization at a temperature of about 42° C. to about 65° C. forup to about 20 hours in a hybridization medium containing 1M NaCl, 50 mMTris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.2%ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 50μg/ml E. coli DNA, followed by washing carried out at between about 42°C. to about 65° C. in a 0.2×SSC buffer.

The BVR protein or polypeptide can also be a fragment of the abovebiliverdin reductase proteins or polypeptides or a variant thereof.

Fragments of BVR preferably contain one or more of the above-listedfunctional domains, and possess one or more of the activities of fulllength BVR. Suitable fragments can be produced by several means.Subclones of a gene encoding a known BVR can be produced usingconventional molecular genetic manipulation for sub cloning genefragments, such as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), and Ausubel et al. (ed.), Current Protocols in MolecularBiology, John Wiley & Sons (New York, N.Y.) (1999 and precedingeditions), each of which is hereby incorporated by reference in itsentirety. The subclones then are expressed in vitro or in vivo inbacterial cells to yield a smaller protein or polypeptide that can betested for a particular activity, e.g., converting biliverdin tobilirubin, modifying cell structure, etc., as discussed infra. See alsoHuang et al., J. Biol. Chem. 264:7844-7849 (1989), which is herebyincorporated by reference in its entirety.

In another approach, based on knowledge of the primary structure of theprotein, fragments of a BVR gene may be synthesized using the PCRtechnique together with specific sets of primers chosen to representparticular portions of the protein. Erlich et al., Science 252:1643-51(1991), which is hereby incorporated by reference in its entirety. Thesecan then be cloned into an appropriate vector for expression of atruncated protein or polypeptide from bacterial cells as describedabove. For example, oligomers of at least about 15 to 20 nt in lengthcan be selected from the nucleic acid molecules of SEQ. ID. No. 2 andSEQ ID. No. 5 for use as primers.

In addition, chemical synthesis can also be employed using techniqueswell known in the chemistry of proteins such as solid phase synthesis(Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964), which is herebyincorporated by reference in its entirety) or synthesis in homogenoussolution (Houbenweyl, Methods of Organic Chemistry, ed. E. Wansch, Vol.15, I and II, Thieme, Stuttgart (1987), which is hereby incorporated byreference in its entirety).

Variants of suitable BVR proteins or polypeptides can also be expressed.Variants may be made by, for example, the deletion, addition, oralteration of amino acids that have either (i) minimal influence oncertain properties, secondary structure, and hydropathic nature of thepolypeptide or (ii) substantial effect on one or more properties of BVR.Variants of BVR can also be fragments of BVR which include one or moredeletion, addition, or alteration of amino acids of the type describedabove. The BVR variant preferably contains a deletion, addition, oralteration of amino acids within one of the above-listed functionaldomains. The substituted or additional amino acids can be either L-aminoacids, D-amino acids, or modified amino acids, preferably L-amino acids.Whether a substitution, addition, or deletion results in modification ofBVR variant activity may depend, at least in part, on whether thealtered amino acid is conserved. Conserved amino acids can be groupedeither by molecular weight or charge and/or polarity of R groups,acidity, basicity, and presence of phenyl groups, as is known in theart.

A number of BVR variants have been described in co-pending U.S. patentapplication Ser. No. 09/606,129 to Maines, filed Jun. 28, 2000, which ishereby incorporated by reference in its entirety.

Variants may also include, for example, a polypeptide conjugated to asignal (or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, identification, ortherapeutic use (i.e., delivery) of the polypeptide.

The BVR protein or polypeptide can be recombinantly produced, isolated,and then purified, if necessary. When recombinantly produced, thebiliverdin reductase protein or polypeptide is expressed in arecombinant host cell, typically, although not exclusively, aprokaryote.

When a prokaryotic host cell is selected for subsequent transformation,the promoter region used to construct the recombinant DNA molecule(i.e., transgene) should be appropriate for the particular host. The DNAsequences of eukaryotic promoters, as described infra for expression ineukaryotic host cells, differ from those of prokaryotic promoters.Eukaryotic promoters and accompanying genetic signals may not berecognized in or may not function in a prokaryotic system, and, further,prokaryotic promoters are not recognized and do not function ineukaryotic cells.

Similarly, translation of mRNA in prokaryotes depends upon the presenceof the proper prokaryotic signals which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference in its entirety.

Promoters vary in their “strength” (i.e., their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operons, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in prokaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ tothe initiation codon (“ATG”) to provide a ribosome binding site. Thus,any SD-ATG combination that can be utilized by host cell ribosomes maybe employed. Such combinations include, but are not limited to, theSD-ATG combination from the cro gene or the N gene of coliphage lambda,or from the E. coli tryptophan E, D, C, B or A genes. Additionally, anySD-ATG combination produced by recombinant DNA or other techniquesinvolving incorporation of synthetic nucleotides may be used.

Mammalian cells can also be used to recombinantly produce BVR orfragments or variants thereof.

Suitable mammalian host cells include, without limitation: COS (e.g.,ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No.CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, andNS-1 cells. Suitable expression vectors for directing expression inmammalian cells generally include a promoter, as well as othertranscription and translation control sequences known in the art. Commonpromoters include, without limitation, SV40, MMTV, metallothionein-1,adenovirus Ela, CMV, immediate early, immunoglobulin heavy chainpromoter and enhancer, and RSV-LTR.

Regardless of the selection of host cell, once the DNA molecule codingfor a biliverdin reductase protein or polypeptide, or fragment orvariant thereof, has been ligated to its appropriate regulatory regionsusing well known molecular cloning techniques, it can then be introducedinto a suitable vector or otherwise introduced directly into a host cellusing transformation protocols well known in the art (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Press, NY (1989), which is hereby incorporated by reference inits entirety).

The recombinant molecule can be introduced into host cells viatransformation, particularly transduction, conjugation, mobilization, orelectroporation. Suitable host cells include, but are not limited to,bacteria, virus, yeast, mammalian cells, insect, plant, and the like.The host cells, when grown in an appropriate medium, are capable ofexpressing the biliverdin reductase, or fragment or variant thereof,which can then be isolated therefrom and, if necessary, purified. TheBVR, or fragment or variant thereof, is preferably produced in purifiedform (preferably at least about 60%, more preferably 80%, pure) byconventional techniques.

For therapeutic purposes, the treated cell is preferably in vivo and theprotein or polypeptide or RNA molecule is delivered into the cell in amanner which affords the protein or polypeptide or RNA molecule to beactive within the cell. A number of known delivery techniques can beutilized for the delivery, into cells, of either proteins orpolypeptides or RNA, or DNA molecules encoding them.

Regardless of the particular method of the present invention which ispracticed, when it is desirable to contact a cell (i.e., to be treated)with a protein or polypeptide or RNA molecule, it is preferred that thecontacting be carried out by delivery of the protein or polypeptide orRNA molecule into the cell.

One approach for delivering protein or polypeptides or RNA moleculesinto cells involves the use of liposomes. Basically, this involvesproviding a liposome which includes that protein or polypeptide or RNAto be delivered, and then contacting the target cell with the liposomeunder conditions effective for delivery of the protein or polypeptide orRNA into the cell.

Liposomes are vesicles comprised of one or more concentrically orderedlipid bilayers which encapsulate an aqueous phase. They are normally notleaky, but can become leaky if a hole or pore occurs in the membrane, ifthe membrane is dissolved or degrades, or if the membrane temperature isincreased to the phase transition temperature. Current methods of drugdelivery via liposomes require that the liposome carrier ultimatelybecome permeable and release the encapsulated drug at the target site.This can be accomplished, for example, in a passive manner wherein theliposome bilayer degrades over time through the action of various agentsin the body. Every liposome composition will have a characteristichalf-life in the circulation or at other sites in the body and, thus, bycontrolling the half-life of the liposome composition, the rate at whichthe bilayer degrades can be somewhat regulated.

In contrast to passive drug release, active drug release involves usingan agent to induce a permeability change in the liposome vesicle.Liposome membranes can be constructed so that they become destabilizedwhen the environment becomes acidic near the liposome membrane (see,e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908(1989), which is hereby incorporated by reference in its entirety). Whenliposomes are endocytosed by a target cell, for example, they can berouted to acidic endosomes which will destabilize the liposome andresult in drug release.

Alternatively, the liposome membrane can be chemically modified suchthat an enzyme is placed as a coating on the membrane which slowlydestabilizes the liposome. Since control of drug release depends on theconcentration of enzyme initially placed in the membrane, there is noreal effective way to modulate or alter drug release to achieve “ondemand” drug delivery. The same problem exists for pH-sensitiveliposomes in that as soon as the liposome vesicle comes into contactwith a target cell, it will be engulfed and a drop in pH will lead todrug release.

This liposome delivery system can also be made to accumulate at a targetorgan, tissue, or cell via active targeting (e.g., by incorporating anantibody or hormone on the surface of the liposomal vehicle). This canbe achieved according to known methods.

Different types of liposomes can be prepared according to Bangham etal., J. Mol. Biol. 13:238-252 (1965); U.S. Pat. No. 5,653,996 to Hsu etal.; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 toHolland et al.; U.S. Pat. No. 5,631,237 to Dzau et al.; and U.S. Pat.No. 5,059,421 to Loughrey et al., each of which is hereby incorporatedby reference in its entirety.

An alternative approach for delivery of proteins or polypeptidesinvolves the conjugation of the desired protein or polypeptide to apolymer that is stabilized to avoid enzymatic degradation of theconjugated protein or polypeptide. Conjugated proteins or polypeptidesof this type are described in U.S. Pat. No. 5,681,811 to Ekwuribe, whichis hereby incorporated by reference in its entirety.

Yet another approach for delivery of proteins or polypeptides involvespreparation of chimeric proteins according to U.S. Pat. No. 5,817,789 toHeartlein et al., which is hereby incorporated by reference in itsentirety. The chimeric protein can include a ligand domain and, e.g.,BVR or a fragment or variant thereof. The ligand domain is specific forreceptors located on a target cell. Thus, when the chimeric protein isdelivered intravenously or otherwise introduced into blood or lymph, thechimeric protein will adsorb to the targeted cell, and the targeted cellwill internalize the chimeric protein.

When it is desirable to achieve heterologous expression of a desirableprotein or polypeptide or RNA molecule in a target cell, DNA moleculesencoding the desired protein or polypeptide or RNA can be delivered intothe cell. Basically, this includes providing a nucleic acid moleculeencoding the protein or polypeptide and then introducing the nucleicacid molecule into the cell under conditions effective to express theprotein or polypeptide or RNA in the cell. Preferably, this is achievedby inserting the nucleic acid molecule into an expression vector beforeit is introduced into the cell.

When transforming mammalian cells for heterologous expression of aprotein or polypeptide, an adenovirus vector can be employed. Adenovirusgene delivery vehicles can be readily prepared and utilized given thedisclosure provided in Berkner, Biotechniques 6:616-627 (1988) andRosenfeld et al., Science 252:431-434 (1991), WO 93/07283, WO 93/06223,and WO 93/07282, each of which is hereby incorporated by reference inits entirety. Adeno-associated viral gene delivery vehicles can beconstructed and used to deliver a gene to cells. The use ofadeno-associated viral gene delivery vehicles in vitro is described inChatterjee et al., Science 258:1485-1488 (1992); Walsh et al., Proc.Nat'l. Acad. Sci. 89:7257-7261 (1992); Walsh et al., J. Clin. Invest.94:1440-1448 (1994); Flotte et al., J. Biol. Chem. 268:3781-3790 (1993);Ponnazhagan et al., J. Exp. Med. 179:733-738 (1994); Miller et al.,Proc. Nat'l Acad. Sci. 91:10183-10187 (1994); Einerhand et al., GeneTher. 2:336-343 (1995); Luo et al., Exp. Hematol. 23:1261-1267 (1995);and Zhou et al., Gene Ther. 3:223-229 (1996), each of which is herebyincorporated by reference in its entirety. In vivo use of these vehiclesis described in Flotte et al., Proc. Nat'l Acad. Sci. 90:10613-10617(1993); and Kaplitt et al., Nature Genet. 8:148-153 (1994), each ofwhich is hereby incorporated by reference in its entirety. Additionaltypes of adenovirus vectors are described in U.S. Pat. No. 6,057,155 toWickham et al.; U.S. Pat. No. 6,033,908 to Bout et al.; U.S. Pat. No.6,001,557 to Wilson et al.; U.S. Pat. No. 5,994,132 to Chamberlain etal.; U.S. Pat. No. 5,981,225 to Kochanek et al.; and U.S. Pat. No.5,885,808 to Spooner et al.; and U.S. Pat. No. 5,871,727 to Curiel, eachof which is hereby incorporated by reference in its entirety.

Retroviral vectors which have been modified to form infectivetransformation systems can also be used to deliver nucleic acid encodinga desired protein or polypeptide or RNA product into a target cell. Onesuch type of retroviral vector is disclosed in U.S. Pat. No. 5,849,586to Kriegler et al., which is hereby incorporated by reference in itsentirety.

Regardless of the type of infective transformation system employed, itcan be targeted for delivery of the nucleic acid to a specific celltype. For example, for delivery of the nucleic acid into specific cells,a high titer of the infective transformation system can be injecteddirectly within the desired site so as to enhance the likelihood of cellinfection within the desired site. The infected cells will then expressthe desired protein product, in this case BVR, or fragments or variantsthereof, to modify the structure of those cells which have beeninfected.

Whether the proteins or polypeptides or nucleic acids are administeredalone or in combination with pharmaceutically or physiologicallyacceptable carriers, excipients, or stabilizers, or in solid or liquidform such as, tablets, capsules, powders, solutions, suspensions, oremulsions, they can be administered orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintranasal instillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, or by application tomucous membranes, such as, that of the nose, throat, and bronchialtubes. For most therapeutic purposes, the proteins or polypeptides ornucleic acids can be administered intravenously.

For injectable dosages, solutions or suspensions of these materials canbe prepared in a physiologically acceptable diluent with apharmaceutical carrier. Such carriers include sterile liquids, such aswater and oils, with or without the addition of a surfactant and otherpharmaceutically and physiologically acceptable carrier, includingadjuvants, excipients or stabilizers. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions.

For use as aerosols, the proteins or polypeptides or nucleic acids insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The materials of the present invention also may beadministered in a non-pressurized form such as in a nebulizer oratomizer.

Both the biliverdin reductase and fragments or variants thereof can bedelivered to the target cells (i.e., at or around the site where cellmodification is desired) using the above-described methods fordelivering such therapeutic products. In delivering the therapeuticproducts to nerve cells in the brain, consideration should be providedto negotiation of the blood-brain barrier. The blood-brain barriertypically prevents many compounds in the blood stream from entering thetissues and fluids of the brain. Nature provides this mechanism toinsure a toxin-free environment for neurologic function. However, italso prevents delivery to the brain of compounds, in this caseneuroprotective compounds that can inhibit nerve cell death following anischemic event.

One approach for negotiating the blood-brain barrier is described inU.S. Pat. No. 5,752,515 to Jolesz et al., which is hereby incorporatedby reference in its entirety. Basically, the blood-brain barrier istemporarily “opened” by targeting a selected location in the brain andapplying ultrasound to induce, in the central nervous system (CNS)tissues and/or fluids at that location, a change detectable by imaging.A protein or polypeptide or RNA molecule of the present invention can bedelivered to the targeted region of the brain while the blood-brainbarrier remains “open,” allowing targeted neuronal cells to uptake thedelivered protein or polypeptide or RNA. At least a portion of the brainin the vicinity of the selected location can be imaged, e.g., viamagnetic resonance imaging, to confirm the location of the change.Alternative approaches for negotiating the blood-brain barrier includechimeric peptides and modified liposome structures which contain a PEGmoiety (reviewed in Pardridge, J. Neurochem. 70:1781-1792 (1998), whichis hereby incorporated by reference in its entirety), as well as osmoticopening (i.e., with bradykinin, mannitol, RPM7, etc.) and directintracerebral infusion (Kroll et al., Neurosurgery 42(5):1083-1100(1998), which is hereby incorporated by reference in its entirety).

Analysis of the promoter region associated with the nucleic acidencoding rBVR indicates the presence of recognition sites for severalregulating proteins, including INF-1, an enhancer of cytokine andvirus-induced transcriptional activation, and AP-1, the proto-oncogenebinding site (McCoubrey et al., “The Structure, Organization andDifferential Expression of the Rat Gene Encoding Biliverdin Reductase,”Gene 160:235-240 (1995), which is hereby incorporated by reference inits entirety. Also, two elements known to be involved in embryonic geneexpression, P3A and engrailed, are present in the promoter region ofthis gene. These criteria are consistent with the function of BVR in aregulatory capacity in the cell.

As discussed in greater detail in the Examples, it has been discoveredthat transformation of mammalian cells with biliverdin reductase iseffective in modifying the structure of the transformed mammalian cells.It is believed that the increase in biliverdin reductase in the cell isresponsible for having modified cell structure. Therefore, one aspect ofthe present invention relates to a method of modifying cell structurewhich includes: increasing the intracellular concentration of biliverdinreductase, or a fragment or variant thereof, in a mammalian cell underconditions effective to modify the structure of the mammalian cell.

Where the cellular concentration of biliverdin reductase is increased,it should be appreciate that some basal level of biliverdin reductasemay exist in the cell which has been targeted. Thus, the increase inbiliverdin reductase intracellular concentration is simply the result ofcausing more biliverdin reductase to be expressed (e.g., inducing ortransforming) or introducing additional biliverdin reductase from anexternal source (i.e., administration).

In contrast, because biliverdin reductase fragments and variants are notnormally expressed in mammalian cells, any increase in biliverdinreductase fragments or variants is the result of their heterologousexpression (i.e., transforming) or introducing biliverdin reductasefragments or variants from an external source (i.e., administering).

Regardless of whether it is biliverdin reductase or its fragments orvariants whose cellular concentration is increased in the mammalian cellto be modified, the increase in concentration can be achieved byintroducing the BVR or BVR fragments or variants into the cell.Typically, this is done by contacting the mammalian cell (to bemodified) with a delivery vehicle which includes biliverdin reductase ora fragment or variant thereof. The delivery vehicle can be any deliveryvehicle of the type described above for protein delivery.

Likewise, such increase in cellular concentration can be achieved byheterologous expression by the mammalian cell (to be modified). Suchheterologous expression is typically the result of transforming themammalian cell with a nucleic acid encoding biliverdin reductase or afragment or variant thereof under conditions effective for expression ofthe biliverdin reductase or the fragment or variant thereof in themammalian cell. The transformation can be achieved using any nucleicacid delivery system of the type described above. (e.g., infectivetransformation).

The mammalian cells which can be treated include, without limitation,stem cells (both omnipotent and pluripotent stem cells), neuronal orglial cells, vascular smooth muscle cells, skeletal muscle cells,epithelial cells, and nucleated blood cells (e.g., macrophages and otherblood cells). The mammalian cells whose structure is modified can beeither in vitro or in vivo when their structure is modified.

Exemplary aspects of the mammalian cell structure which can be modifiedin accordance with the present invention include, without limitation,enhanced cell size (i.e., forming giant cells), actin microspikeformation, polar cell morphology (i.e., with protracted filopodiaextensions), and a combination thereof.

Without being bound by theory, it is believed that the modified cellstructure is the result of biliverdin reductase interaction withproteins and kinases that govern cell cycling and with polypeptidegrowth factors.

In view of the modified cell structure, it is further contemplated thatthe present invention can be utilized to perform organogenesis, tissueremodeling, wound healing, angiogenesis, or combinations thereof. Tissueremodeling, of course, encompasses both wound healing and angiogenesis.

Thus, a further aspect of the present invention relates to a method ofperforming in vivo tissue remodeling in a mammal. This aspect of theinvention includes: delivering biliverdin reductase, or fragments orvariants thereof, to one or more cells present at a site of tissueremodeling in a mammal, wherein the delivering (of BVR or its fragmentsor variants) increases the intracellular concentration of biliverdinreductase, or fragments or variants thereof, under conditions effectiveto modify the structure of the one or more cells at the site of tissueremodeling, thereby remodeling the tissue containing the one or morecells.

Tissues which can be remodeled in vivo include, without limitation,epithelial tissues, nerve tissues, muscular tissues (both smooth muscleand skeletal muscle tissues), or connective tissue. More specifically,angiogenesis can implicate remodeling of vascular tissue and modifyingthe structure of vascular smooth muscle, bladder, and urinary tractcells. Likewise, wound healing can implicate remodeling of epithelialtissues, nerve tissues, muscular tissues (both smooth muscle andskeletal muscle tissues), or connective tissues via modifying thestructures of epithelial cells, nerve or glial cells, vascular andskeletal muscle cells, etc.

As a result of such tissue remodeling, where multiple tissues areremodeled, it also contemplated to utilize the present inventionaccording to a method of repairing a damaged organ or organ system byperforming the method of in vivo tissue remodeling in accordance withthe present invention, where the site of tissue remodeling is within thedamaged organ or organ system. Exemplary organ or organ systems whichcan be subject to repair include, without limitation, skin, liver,nervous system (e.g., both sensory neurons and motor neurons),cardiovascular system, and urogenital tract.

With respect specifically to wound healing, it should be appreciatedthat the primary goal in the treatment of wounds is to achieve woundclosure. Open cutaneous wounds represent one major category of woundsand include burn wounds, neuropathic ulcers, pressure sores, venousstasis ulcers, and diabetic ulcers. Open cutaneous wounds routinely healby a process which comprises six major components: i) inflammation, ii)fibroblast proliferation, iii) blood vessel proliferation, iv)connective tissue synthesis v) epithelialization, and vi) woundcontraction. Wound healing is impaired when these components, eitherindividually or as a whole, do not function properly. Numerous factorscan affect wound healing, including malnutrition, infection,pharmacological agents (e.g., actinomycin and steroids), diabetes, andadvanced age (see Hunt and Goodson, 1988). In general, agents whichpromote a more rapid influx of fibroblasts, endothelial and epithelialcells into wounds should increase the rate at which wounds heal. Byvirtue of increasing the intracellular concentration of biliverdinreductase, it becomes possible to induce an increase in cell size, theformation of actin microspikes, and morphological changes in cellpolarity, i.e., formation of filopodia extensions. These aspects suggestthat the affected cells can be made more readily able to influx intodamage sites in need of repair.

The use of BVR for wound healing can also be carried out in combinationwith a medicament selected from the group consisting of an antibacterialagent, an antiviral agent, an antifungal agent, an antiparasitic agent,an anti-inflammatory agent, an analgesic agent, an antipruritic agent,or a combination thereof For cutaneous wound healing, a preferred modeof administration is by the topical route.

EXAMPLES

The following examples are intended to illustrate, but by no means areintended to limit, the scope of the present invention as set forth inthe appended claims.

Example 1 In Vitro Transformation of HeLa Cells with BiliverdinReductase for Modifying Cellular Structure

HeLa cells were transfected in vitro with biliverdin reductase encodingDNA. A HeLa cell suspension having a density of about 12×10⁴/ml wasintroduced to a 12-well plate using 0.2 ml of the cell suspension perwell (i.e., about 2.4×10⁴ cells per well). The following protocol wasemployed for transfection:

Cells were washed with DMEM (serum). Thereafter, the following solutionwas added: 2 μl of DNA (541 0.5 μg/μl), 50 μl of DMEM(−), and 2 μl oflipofectimine. After 4-5 h, 0.5 ml of DMEM (20% serum) was addedfollowed by 30 h incubation (37° C.).

On the following day cells were immunostained using the followingprotocol. HeLa cells were washed once for 5 min in PBS (0.1% PB, 0.9%NaCl), followed by treatment with 4% PFA (on ice). 10 min later, cellswere washed 3 times for 5 min each time in PBS. Cells were blocked bytreatment with PBS (950 μl)+50 μl horse serum (5% HS PBS) for 1 h atroom temperature. Cells were treated with a solution of 3% HS-0.25%Triton-PBS at 4° C. overnight, thereafter cells were treated with 1:1000dilution of BVR antibody.

For antibody staining, cells were washed 3 times for 5 min each time inPBS—0.25% Triton×100 and treated with second antibody solutionconsisting of: horse serum, 15 μl/ml and antimouse-IgG, 5 μl/ml. After 3times washing with PBS for 5 min each time, cells were visualized usingABC solution (Vector Labs) and stained for 30 min.

Non specific staining was removed using 3% of H₂O₂ . Cells were thenwashed with dd H₂O and dehydrated in 95%-100% ethanol, xylene 5 min.Slides were mounted with ½ permount+½ xylene

Control and transformed HeLa cells were visualized by immunostainingusing antibody to BVR. As shown in FIGS. 1-6, the transformed HeLa cellsdisplayed larger cell size relative to control cells, formation of actinmicrospikes, and polar cell morphology with filopodia extensions.

The above results indicate that BVR is a regulator of cell proliferationand cell differentiation. Following transformation to induce an increasein BVR expression, transfected cells were transformed into giant cellsseveral times the size of normal cells. Moreover, the transfected cellsdisplayed formation of actin microspikes. Such actin microspikes areknown to act as sensory devices by which cells explore theirenvironment. Also, BVR transfected cells exhibited polar cellmorphology, as characterized by protracted filopodia extensions thatresemble that of neuronal axon and dendritic extensions, a phenotypewhich is not displayed by Cdc42 transfected cells (Adams and Schwarz,“Stimulation of Fasein Spikes by Thrombospondin-1 is Mediated by GTPasesRac and Cdc42,” J. Cell Biol. 150:807-822 (2000), which is herebyincorporated by reference in its entirety). These properties aredisplayed by certain cyclin-dependent kinases. Specifically, Cdc42kinase stimulates spike formation (Kozma et al., “The Ras-relatedprotein Cdc42Hs and Bradykinin Promote Formation of Peripheral ActinMicrospikes and Filopodia in Swiss 3T3 Fibroblasts,” Mol. Cell Biol.15:1942-1952 (1995); Nobes and Hall, “Rho, Rac, and Cdc42 GTPasesRegulate the Assembly of Multimolecular Focal Complexes Associated withActin Stress Fibers, Lamellipodia, and Filopodia,” Cell 81:53-62 (1995);Adams and Schwarz, “Stimulation of Fasein Spikes by Thrombospondin-1 isMediated by GTPases Rac and Cdc42,” J. Cell Biol. 150:807-822 (2000),each of which is hereby incorporated by reference in its entirety) and Dtype cyclins, e.g., cyclin D₂, deregulate cell size and cause cell massincrease (Kershoff and Ziff, “Cyclin D₂ Ha-Ras Transformed Rat EmbryoFibroblasts Exhibit Novel Deregulation of Cell Size Control and Early SPhase Arrest in Low Serum,” EMBO J. 14:1892-1903 (1995), which is herebyincorporated by reference in its entirety). These proteins requirecooperation of signal transduction kinase activity, e.g., GTPasesRac/Ha-Ras (Adams and Schwarz, “Stimulation of Fasein Spikes byThrombospondin-1 is Mediated by GTPases Rac and Cdc42,” J. Cell Biol.150:807-822 (2000); Kershoff and Ziff, “Cyclin D₂ and Ha-Ras TransformedRat Embryo Fibroblasts Exhibit Novel Deregulation of Cell Size Controland Early S Phase Arrest in Low Serum,” EMBO J. 14:1892-1903 (1995),each of which is hereby incorporated by reference in its entirety). BVR,as noted above, is a protein kinase and has both cell proliferating andcell differentiation activities. Furthermore, BVR can unexpectedlycontrol the cell size under normal conditions, whereas cyclin D₂ andHa-Ras transformed cells only display giant size in low serumconditions. The morphology of the above-described transformed cells isalso consistent with the use of BVR expression for promoting axonalgrowth in the case of nerve damage.

In a number of amino acid sequences, X is used to depict a residue whichcan be any naturally occurring amino acid, unless otherwise indicated.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method of in vivo tissue remodeling in a mammal comprising:delivering biliverdin reductase, or fragments or variants thereof, toone or more cells present at a site of tissue remodeling in a mammal,wherein said delivering increases the intracellular concentration ofbiliverdin reductase, or fragments or variants thereof, under conditionseffective to modify the structure of the one or more cells at the siteof tissue remodeling, thereby remodeling the tissue containing the oneor more cells.
 2. The method according to claim 1, wherein the tissue isepithelial tissue, nerve tissue, muscular tissue, or connective tissue.3. The method according to claim 1 wherein the one or more cells is astem cell, a neuronal or glial cell, a vascular smooth muscle cell, askeletal muscle cell, an epithelial cell, a nucleated blood cell, or acombination thereof.
 4. The method according claim 1 wherein saiddelivering comprises: introducing biliverdin reductase into the one ormore cells.
 5. The method according claim 4 wherein said introducingcomprises: contacting each of the one or more cells with a deliveryvehicle comprising biliverdin reductase.
 6. The method according toclaim 5 wherein the delivery vehicle comprises a fusion proteincomprising biliverdin reductase and a ligand domain recognized by areceptor of the one or more cells.
 7. The method according to claim 5wherein the delivery vehicle comprises a liposome containing biliverdinreductase.
 8. The method according to claim 5 wherein the deliveryvehicle comprises an enzymatically stable conjugate comprising a polymerand biliverdin reductase conjugated to the polymer.
 9. The methodaccording to claim 1 wherein said delivering comprises: transformingeach of the one or more cells with a nucleic acid encoding biliverdinreductase under conditions effective for expression of the biliverdinreductase in the one or more cells.
 10. The method according to claim 9wherein said transforming comprises: transfecting each of the one ormore cells with an infective transformation vector comprising thenucleic acid encoding biliverdin reductase.
 11. The method according toclaim 10 wherein the infective transformation vector is an adenovirusvector or a retrovirus vector.
 12. A method of repairing a damaged organor organ system by performing said method of in vivo tissue remodelingaccording to claim 1, where the site of tissue remodeling is within thedamaged organ or organ system.
 13. The method according to claim 12,wherein the organ or organ system is skin, liver, nervous system,cardiovascular system, or urogenital tract.